The lipoproteins of human blood can perform a series of various functions. A well studied and well known function of lipoproteins is the transport of lipids. Lipoproteins have the ability to bind insoluble lipids, to transport them in an aqueous milieu, and bring them to their destination. For some time the individual classes of lipoproteins have been studied more closely in connection with disturbances of lipid metabolism. In the majority of Western countries a positive correlation between cardiovascular diseases and high plasma cholesterol or high low density lipoprotein cholesterol (LDL cholesterol), and a negative correlation to high HDL or high HDL cholesterol was shown by epidemiological studies. Although a whole range of medicines is available on the market which have been shown to have a lipid-lowering effect, it is conceivable that in certain cases a substitution of suitable lipoproteins is advisable. Suitable in such cases are, first of all, the "good" lipoproteins such as HDL or HDL-like particles, such as reconstituted HDL (rHDL) from isolated apolipoproteins and suitable lipids.
Besides the lipids, which are ingested from food, lipoproteins from other lipids or lipoidal substances can be transported or bound. For example, components of dead cells can be bound to lipoproteins and can be given a new function. Lipoidal substances can also be bound to lipoproteins, such as, for example, lipopolysaccharide (LPS). Lipopolysaccharides or endotoxins are components of membranes of gram-negative bacteria. If sufficiently large quantities of endotoxin are able to enter the cardiovascular system, this can lead to septic shock or even death. As a result of binding of LPS to lipoproteins, the function or activity of LPS can be modulated. The activity of LPS can be considerably changed in vivo or in vitro through addition of lipoproteins or lipoprotein-like particles. Thus it could be shown in vivo that the addition of reconstituted HDL inhibited the formation of tumor necrosis factor (TNF), an important mediator of sepsis. In vivo, the symptoms of shock could be greatly reduced through the administration of HDL or rHDL.
Besides interactions of lipoproteins or reconstituted lipoproteins with lipids or lipoidal substances, Interactions of lipoproteins with proteins have also been described:
lipoproteins can enter into interactions with individual components of the complement system, and can therefore influence their activity; PA1 components of the coagulation systems are likewise known which are to be found in the lipoprotein fraction, that is, which are associated with certain lipoproteins; PA1 acute phase proteins such as serum amyloid A (SAA) are found in the HDL fraction; PA1 and furthermore the adsorption of certain proteins on surfaces can be influenced by pretreatment with lipoproteins. PA1 The platelets' ability to be activated can be reduced through binding of HDL or can be stimulated by addition of LDL; PA1 Monocytes and macrophages likewise have receptors for lipoproteins; the binding or uptake of lipoproteins can lead to changes in the activities of these cells; PA1 The activity of other cells involved in the host defense system such as neutrophils can likewise be modified or modulated through binding of lipoproteins; PA1 Furthermore, the growth of tumor cells, shown using glyoblastoma cells as an example, can also be influenced through lipoproteins. PA1 high dilutions of solutions in intermediate products make processing of the mixtures in the required time impossible; PA1 the necessary infrastructure for large volumes is normally not available; PA1 the organic solvents used are not environmentally acceptable; PA1 the final products cannot be stored; PA1 the concentration of the final product is too low, thus the volumes to be infused in the patient would be too large; PA1 the products normally have to be purified further, for example by means of gel chromatography to separate residual free lipid and/or free protein from the desired rHDL particles.
Through specific or non-specific binding to cells, lipoproteins can also influence cellular activity:
In scientific literature reports are to be found moreover which describe interactions of lipoproteins with pathogens; for example, an antimicrobial activity is ascribed to lipoproteins. It has been shown that viruses can also be inactivated by means of lipoproteins, or, using trypanosomes as an example, parasites can be influenced or respectively inhibited.
These examples show diverse possibilities for the use of lipoproteins or rHDL in prophylactic and therapeutic applications.
Lipoproteins are divided into four main classes:
chylomicrons, which are particles that consist predominantly of triglycerides and which normally appear in larger quantities in plasma only after fatty meals, VLDL (Very Low Density Lipoproteins), LDL (Low Density Lipoproteins), and HDL (High Density Proteins). The nomenclature arises from the isolation of lipoproteins by means of ultracentrifugation. Classically, the lipoproteins are isolated in a density gradient. This method can only be applied on a laboratory scale as it requires a specialized apparatus and is very time-consuming. At best only a few hundred milligrams of lipoproteins or apolipoproteins can be produced using this method over a course of a few days. Other methods of isolating apolipoproteins or lipoproteins have also been known for some time; they are based in many cases on precipitation by means of divalent cations and/or polyethylene glycol or dextran, for example. A further possibility of isolating apolipoproteins is precipitation by means of alcohol fractionation, as described in patent EP 0 329 605 B1. Using this lasts mentioned method it is possible to isolate larger quantities of apolipoprotein A-I (apoA-I) or fractions which are enriched in apoA-I, and to make them available for therapeutic applications. Using thus isolated apoA-I or protein fractions enriched in apoA-I, a series of experiments have been carried out, both with animals and with humans. Based on these experiments, both the safety of these products with respect to viruses could be shown and also that apoA-I in the form used leads to no significant side reactions in humans or animals. Nevertheless, in in vitro tests none of the desired activities could be found such as, for example, cholesterol transport or effects of apoA-I on cells such as neutrophils, macrophages, monocytes or platelets. In in vivo tests both in animals and in humans very short half-lives of apoA-I in plasma were observed. Because of the molecular weight of free apoA-I (28 000 Dalton), there exists the possibility that apoA-I is eliminated by the kidneys. apoA-I could in fact be detected in the urine of rabbits. These results demonstrate that apoA-I infused in large quantities is not distributed to the desired lipoprotein fraction. Due to its short half-life, apoA-I is able to have its effect in vivo at most for a very short time. Consequently a more suitable form of administration is achieved by infusing apoA-I in a lipoprotein or a lipoprotein-like particle.
Methods of producing reconstituted lipoproteins have been described in scientific literature, especially for apolipoproteins A-I, A-II, A-IV, apoC and apoE (A. Jonas, Methods in Enzymology 128, 553-582 (1986)). The is most frequent lipid used for reconstitution is phosphatidyl choline, extracted either from eggs or soybeans. Other phospholipids are also used, also lipids such as triglycerides or cholesterol. For reconstitution the lipids are first dissolved in an organic solvent, which is subsequently evaporated under nitrogen. In this method the lipid is bound in a thin film to a glass wall. Afterwards the apolipoprotein and a detergent, normally sodium cholate, are added and mixed. The added sodium cholate causes a dispersion of the lipid. After a suitable incubation period, the mixture is dialyzed against large quantities of buffer for a longer period of time; the sodium cholate is thereby removed for the most part, and at the same time lipids and apolipoproteins spontaneously form themselves into lipoproteins or so-called reconstituted lipoproteins. As alternatives to dialysis, hydrophobic adsorbents are available which can adsorb detergents (Bio-Beads SM-2, Bio Rad; Amberlite XAD-2, Rohm & Haas) (E. A. Bonomo, J. B. Swaney, J. Lipid Res., 29, 380-384 (1988)), or the detergent can be removed by means of gel chromatography (Sephadex G-25, Pharmacia). Lipoproteins can also be produced without detergents, for example through incubation of an aqueous suspension of a suitable lipid with apolipoproteins, the addition of lipid which was dissolved in an organic solvent, to apolipoproteins, with or without additional heating of this mixture, or through treatment of an apoA-I-lipid-mixture with ultrasound. With these methods, starting, for example, with apoA-I and phosphatidyl choline, disk-shaped particles can be obtained which correspond to lipoproteins in their nascent state. Normally, following the incubation, unbound apolipoprotein and free lipid are separated by means of centrifugation or gel chromatography in order to isolate the homogeneous, reconstituted lipoprotein particles.
Described in U.S. Pat. No. 5,128,318 is a method of producing reconstituted HDL wherein phosphatidyl choline is dissolved in a solution with the aid of an organic solvent.
The methods for producing reconstituted lipoproteins described above are only suitable for smaller quantities of some milligrams to at most some grams on the laboratory scale:
Furthermore described in A. Hubsch et al., Circulatory Shock 40, 14-23 (1993) is a method of producing reconstituted lipoproteins, wherein a ratio of apolipoprotein to lipid of 1:200 is used. The result of this procedure is that the product obtained has a considerable portion of free lipid, which unfavorably influences its therapeutic applicability.
For the therapeutic or prophylactic use of rHDL in humans, a dose of rHDL in gram quantities is necessary to achieve significant increases of the apoA-I or HDL level in the plasma. Thus, the economical production of rHDL for clinical purposes on a kilogram or larger scale is not possible with the methods described above.